Project Summary/Abstract Background: The discovery of new drug leads is a constant biomedical need. Following a recently developed metabologenomics approach, the new anti-cancer actinobacterial secondary metabolite tambromycin was discovered and correlated to a biosynthetic gene cluster (BGC). Tambromycin is a novel non-ribosomal peptide which contains a new pyrrolidine-containing amino acid named tambroline. Tambromycin shares some structural features with the known metabolites JBIR-34 and JBIR-35. High sequence similarity was detected between genes encoding for similar non-ribosomal peptide synthetase (NRPS) enzymes in the biosynthetic gene clusters for tambromycin and JBIR-34/35. However, the biogenesis of the new tambroline unit remain unclear. Also, while tambromycin possesses anti-cancer properties, JBIR-34/35 (which lack tambroline) have no reported cytotoxicity suggesting that tambroline is required for biological activity. The proposed studies will give definitive answers to the ambiguous steps in tambromycin biosynthesis, will elucidate its intracellular binding target, and will lead genome mining for similar novel metabolites. Specific Aims: 1) Study the biogenesis and incorporation of the new amino acid unit tambroline; 2) Target Identification of tambromycin via chemical proteomics; 3) Tambroline- based genome mining. Research Design and Methods: Two acyl-CoA dehydrogenases (ACADs) are suggested to be involved in tambroline biosynthesis from lysine. Feeding experiments using stable isotope labeled lysine provided preliminary evidence for the proposed mechanism. To test this hypothesis further, the ACAD enzymes will be cloned and heterologously expressed to reconstitute the biosynthesis of tambroline in vitro. Moreover, an unassigned NRPS modules in the tambromycin gene cluster is hypothesized to be capable of adenylating and loading tambroline after its production through pre-assembly line processing. Accordingly, the A-T didomain from the candidate NRPS protein will be cloned, expressed and incubated with potential substrates, and the relative ability to adenylate amino acids will be evaluated. Gene knockout experiments will be tackled for additional confirmation. Additionally, biotinylated molecular probes will be prepared and utilized in a protein pull-down assay and the proteins will be identified by high-performance proteomics via LC-MS, in order to identify the binding target of tambromycin. Two other natural products, azinomycin B and ficellomycin, comprise different pyrrolidine-containing amino acid substructures and have also demonstrated anti-cancer activities. By examining the BGCs of tambromycin and azinomycin B for common features, a biosynthetic cassette for pyrrolidine-containing amino acids was proposed. This cassette will be used for genome mining in different actinomycete strains to identify BGCs with novel pyrrolidine-containing natural products. Metabologenomics data will be explored for new compounds with high correlation scores to these mined BGCs. Chemical hits will be isolated, chemically and biosynthetically characterized and tested for biological activities.